Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells

Significance Studying the functions of G-protein-coupled muscarinic receptors, which are protein tyrosine kinase independent, might inspire new cancer therapies by bypassing classical TCR signaling pathways. Stimulating heterologously expressed muscarinic receptors (M1 and the synthetic hM3Dq) induced calcium responses and phosphorylation of ERK in preactivated T cells if PLCβ1 was coexpressed. Unexpectedly, whereas stimulation of hM3Dq that couples to PLCβ1 induced high IFN-γ, CD69, and CD25 expression, surprisingly, it did not induce high IL-2 expression. Stimulation of hM3Dq reduced IL-2 mRNA stability which correlated with an effect on the IL-2 mRNA stability. The selective effect on IL-2 mRNA may be attributable to reduced pAKT downstream pathway function, suggesting that the pAKT pathway is critical for IL-2 production.

and Go proteins (10). Stimulation of M1, M3, and M5 receptor subtypes activates Gq proteins, leading to activation of the membrane-bound phospholipase Cβ (PLCβ) enzymes (9). This results in increases in cytoplasmic free calcium concentrations and activation of PKC and the Ras/MAPK pathways (9,10), signaling pathways critically important for the induction of numerous cytokines and other events associated with T cell activation. We have previously shown that the stimulation of the human M1 receptor (hM1) bypasses PTK-dependent TCR pathways to induce calcium increases, ERK activation, and IL-2 production in the human Jurkat T cell leukemic line (6)(7)(8).
To study the biochemical signaling and functional consequences of stimulating muscarinic receptors in primary T cells, we used two muscarinic receptors: 1) the murine M1 receptor, which is activated by the native ligand acetylcholine or by a pharmacologically agonist carbachol, and 2) an engineered human M3 receptor named Gq-coupled hM3 DREADD "hM3Dq" (DREADD: designer receptors exclusively activated by designer drugs), which can be selectively activated by the normally physiologically inert synthetic ligand clozapine (11,12). Using this system, we found that high-level expression of PLCβ1 in T cells was required, as was high-level expression of the muscarinic receptors, for effective signaling. Stimulation of the muscarinic receptors induced many of the events associated with T cell activation via the TCR. However, we also identified an unanticipated requirement for AKT pathway activity for IL-2 production but not for several other cytokines, as well as an inhibitory effect of the muscarinic receptors on the AKT pathway.

PLCβ1 Greatly Enhances Calcium Responses by Activated
Muscarinic Receptors in Primary T Cells. We first studied the biochemical signaling and functions of two types of muscarinic receptors, M1 (flag epitope tagged) and hM3Dq (HA epitope tagged), which can be stimulated by carbachol or clozapine, respectively, in Jurkat leukemic T cells. Transduction of Jurkat cells with lentiviruses containing either muscarinic receptor (M1 or hM3Dq) led to results that were consistent with our previous findings (6,7), namely, that stimulation of muscarinic receptors (M1 or hM3Dq) induced strong calcium responses in Jurkat cells ( Fig. 1 A and B). Muscarinic receptors were then virally transduced into primary mouse T cells to determine whether their stimulation can induce signaling in these cells. Retroviral transduction of either muscarinic receptor (M1 or hM3Dq) in primary mouse T cells showed that both of these receptors were responsive to stimulation with carbachol or clozapine, respectively, at least with regard to calcium increases ( Fig. 1 C and D). However, the magnitudes of these responses were much lower than those observed in Jurkat cells ( Fig. 1 A-D). PLCβ, downstream of Gq, is activated by muscarinic receptor stimulation. The predominant isoforms expressed in Jurkat cells are the PLCβ1, 2, and 3 isoforms (13) (SI Appendix, Fig. S1A). Neuronal cells, which naturally express different types of muscarinic receptors (14), exhibited the highest expression of PLCβ1 among all PLCβ isoforms (SI Appendix, Fig. S1B). In contrast, mouse-naïve or activated CD4 and CD8 T cells did not express PLCβ1 either at the mRNA and protein levels (SI Appendix, Fig. S1 C-F). The predominant PLCβ isoforms expressed in primary human and mouse CD4 and CD8 T lymphocytes are PLCβ2 and PLCβ3 (13,15) (SI Appendix, Fig. S1 C-F). Significantly, PLCβ1 is important in mediating optimal IL-2 production in response to superantigens in Jurkat cells (16). Therefore, we determined whether transduction of PLCβ1 in mouse T cells could improve responses to stimulated muscarinic receptors in mouse T cells. Fortunately, viral transduction of PLCβ1 strongly amplified the induced calcium responses of either of the stimulated muscarinic receptors ( Fig. 1 C and D). Thus, the transduced PLCβ1 appeared to more efficiently mediate signals from the stimulated transduced muscarinic receptors than the endogenously expressed PLCβ isoforms.

hM3Dq+ PLCβ1+ (hM3Dq/β1) T Cells Preactivated by TCR and CD28 Stimulation Exhibited Large Calcium and pErk Responses
to hM3Dq Agonist. We generated mice with the properly inserted PLCβ1cDNA in the Rosa 26 locus (SI Appendix, Fig. S2 A and B). The expression of this PLCβ1 transgene in T cells was validated by protein blot analysis (SI Appendix, Fig. S2C) and could be monitored by the expression of the fluorescent protein mCherry separated from PLCβ1 by an IRES sequence (SI Appendix, Fig. S2 A and B). Inducible PLCβ1 knock-in mice were crossed with hM3Dq x Lck-Cre mice to generate mice in which only T cells express both hM3Dq and PLCβ1 to study the muscarinic receptor-regulated activation of PLCβ1 in T cells (SI Appendix, Fig. S2  have the transgene integrated on chromosome 14, which has not adversely affected the functionality of the allele (https://www.jax. org/strain/026220). Lck Cre-mediated removal of an upstream floxed-STOP cassette allows hM3Dq-mcitrine and PLCβ1mcherry to be expressed in T cells in Lck-Cre-hM3Dq-PLCβ1 mice. All subsequent experiments used only Lck-Cre-hM3Dq-PLCβ1 T cells that expressed both hM3Dq and PLCβ1. For simplicity, we labeled these cells as hM3Dq/β1 T cells. Thymocytes showed increased expression of hM3Dq and PLCβ1 from CD4SP and CD8SP relative to the amounts expressed in DP thymocytes ( Fig. 2A). DP and SP thymocytes from Lck-Cre-hM3Dq-PLCβ1 mice exhibited weak calcium responses to clozapine stimulation (Fig. 2B). Surprisingly, even though peripheral hM3Dq/β1 T cells expressed hM3Dq and PLCβ1, these T cells showed no calcium responses to clozapine stimulation ( Fig. 2 C and D). However, we found that hM3Dq/β1 T cells prestimulated with anti-CD3 and anti-CD28 mAbs and cultured for 3 d and then incubated with either IL-2 or IL-7 for 2 d showed markedly increased calcium responses to clozapine stimulation (Fig. 2E). This might be attributable to the higher PLCβ1 and hM3Dq expression in T cells stimulated with anti-TCR/CD28 reactive mAbs and IL2 ( Fig. 2F and SI Appendix, Fig. S2F). Indeed, the preactivated T cells with more highly expressed hM3Dq and PLCβ1 exhibited larger calcium responses compared to those with lower hM3Dq and PLCβ1 expression (SI Appendix, Fig. S2G). Thus, TCR/ CD28 stimulation and IL-2 (or IL-7) incubation led to larger calcium increases in hM3Dq/β1 T cells in response to clozapine stimulation most likely due to the increased expressions of hM3Dq and PLCβ1. These preactivated hM3Dq/β1 T cells were used in upcoming experiments to study the functional consequences of activating the muscarinic receptors in primary T cells. Stimulation of hM3Dq or M1 receptors that could couple to PLCβ1 also induced larger and more sustained calcium and pErk responses ( Fig. 2 G and H and SI Appendix, Fig. S2 H-K). In summary, activation of hM3Dq which was able to couple to PLCβ1 induced substantial calcium increases and phosphorylated ERK responses in preactivated primary T cells.
In Jurkat cells, muscarinic receptors bypass PTK-dependent TCR signaling pathways (6)(7)(8). Similarly, the Src kinase inhibitor PP2 suppressed induced phosphotyrosine, pERK, and calcium responses by anti-CD3 stimulation but only showed minimal effects in pERK and calcium responses to clozapine stimulation in hM3Dq/β1 T cells ( Fig. 2 I-M). Thus, these results suggested that muscarinic receptors can bypass classical PTK-dependent TCR pathways in primary T cells.
Stimulation of hM3Dq Induces IFN-γ but Reduces IL-2 in TCR-Stimulated hM3Dq/β1 T Cells. Stimulation of muscarinic receptors with clozapine did not induce the expression of IL-2, but potently induced IFN-γ, CD69, and CD25 expression in hM3Dq/β1 CD4 T cells (Fig. 3 A-H). Interestingly, addition of clozapine to TCRstimulated T cells resulted in significantly reduced IL-2 responses but did not change or in some cases even increased IFN-γ, CD69, and CD25 expression in hM3Dq/β1 CD4 T cells (Fig. 3 A-H). We also observed similar effects in hM3Dq/β1 CD8 T cells (SI Appendix, Fig. S3 A-H). Previously, costimulation with PMA plus carbachol was reported to lead to increased IL-2 production in hM1+ Jurkat cells (7). However, clozapine stimulation combined with PMA or CD28 did not induce IL-2, nor did it increase IFNγ and CD25 responses in primary hM3Dq/β1 T cells compared with clozapine alone (Fig. 3 I-P). Stimulation with clozapine and PMA led to increased CD69 expression compared with clozapine alone (Fig. 3 K and O). Similar to TCR stimulation, addition of clozapine also resulted in reduced IL-2, but not IFN-γ in CD28-or PMA-stimulated hM3Dq/β1 T cells (Fig. 3 I-P). In summary, stimulation of the hM3Dq receptor with clozapine with or without costimulation (PMA or anti-CD28) induces IFN-γ, CD69, and CD25. However, stimulation with clozapine does not substantially increase IL-2 and, instead, leads to decreased IL-2 expression in TCR costimulated hM3Dq/β1 T cells.

Stimulation of hM3Dq Induces Nuclear Translocation of NFAT
and NFκB and Activates AP-1 in hM3Dq/β1 T Cells. Since nuclear factor of activated T cells (NFAT) family members, nuclear factor-κB (NFκB), and activator protein 1 (AP-1) bind to the promoter and upstream regions relative to the transcription start site of IL-2, we determined whether activation of hM3Dq receptors induced activation of these transcription factors. We found that clozapine induced nuclear translocation of NFAT and NFκB and also phosphorylated c-Jun (an AP-1 subunit) in preactivated hM3Dq/β1 CD4 T cells (Fig. 4 A and B). Flow cytometry of nuclear staining for NFAT and NFκB1 consistently confirmed that stimulation of hM3Dq greatly induced nuclear translocation of NFAT and NFκB, comparably to PMA + ionomycin or to TCR stimulation, albeit with somewhat altered kinetics ( Fig. 4 C-F). In summary, stimulation of muscarinic receptors with PLCβ1 induced strong nuclear translocation of NFAT and NFκB and phorphorylated c-Jun, an indication of AP-1 activation, in hM3Dq/β1 T cells.

Stimulation of hM3Dq Results in Reduced IL-2 mRNA Stability Measured by 3′UTR Activity of IL-2 mRNA in TCR-Prestimulated
hM3Dq/β1 CD4 T Cells. To determine whether clozapine stimulation differentially affects cytokine mRNA expression, we measured various cytokine mRNA at 4 and 8 h after stimulation. Compared to stimulation of the TCR, stimulation of muscarinic receptors resulted in smaller increases in IL-2 mRNA, but larger relative increases in IFNγ, IL-4, and IL-13 mRNA in hM3Dq/ β1 CD4 T cells ( Fig. 5 A-D). Thus, these results are consistent with IL-2 and IFNγ protein expression detected by intracellular staining (Fig. 3 A, B, E, and F), and cells stimulated via muscarinic receptors exhibited greatly reduced IL-2 but increased IFNγ, IL-4, and IL-13 mRNA expression in TCR-prestimulated hM3Dq/β1 CD4 T cells at 4 h after stimulation ( Fig. 5 A-D).
IL-2 mRNA stability is highly regulated by RNA-binding proteins that target the 3′ untranslated mRNA segment (18). To determine whether the differential effects of clozapine on IL-2 versus IFN-γ expression in TCR-stimulated hM3Dq/β1 CD4 T cells might be due to a differential RNA stability that was governed by the 3′ untranslated region, we transfected the cells with the Psicheck2 plasmid in which Renilla luciferase reporter transcript contains 3′UTRs of either IL-2 or IFN-γ mRNA and performed luciferase assays ( Fig. 5 E and F). Our results showed that the reporter carrying a 3′UTR derived from IL-2 but not the IFN-γ mRNA was strongly reduced in the TCR plus clozapine stimulation compared to TCR stimulation alone in hM3Dq/β1 CD4 T cells (Fig. 5 E and F). Thus, stimulation of muscarinic receptor resulted in an active reduction in IL-2 mRNA stability by reducing 3′UTR activities of IL-2 mRNA but showed no effects in 3′UTR activities of IFN-γ mRNA in TCR-stimulated hM3Dq/β1 CD4 T cells. In summary, addition of clozapine stimulation of the hM3Dq results in reduced 3′UTR activities of IL-2 mRNA, which can at least partially explain the potent decrease in IL-2 mRNA and protein expression in clozapine plus TCR-stimulated hM3Dq/β1 CD4 T cells.  clozapine negatively impacted proximal TCR signaling pathways in hM3Dq/β1 CD4 T cells. Interestingly, our results showed that costimulation of hM3Dq did not affect major proximal TCR signaling events that induce tyrosine phosphorylation of various tyrosine phosphoproteins downstream of the TCR (Fig. 6A). However, since signaling through the PI3K on T cells can modulate the half-life of a select subset of cytokine mRNAs, such as IL-2 via RNA-binding proteins (RBPs) (19,20), we assessed whether clozapine affects PI3K signaling pathway in stimulated T cells. Activation of PI3K leads to production of PIP3 at the plasma membrane (21,22). Cytosolic AKT is recruited to the membrane and engages PIP3 through its PH domain and is phosphorylated at T308 and S473 by PDK1 and mTORC2, respectively (21,23). Stimulation of muscarinic receptors greatly reduced activation of pAKT both at S473 and T308 in anti-CD3-stimulated hM3Dq/β1 T cells (Fig. 6B). Clozapine only showed small decreases or no effects in phosphorylated PI3K and negative regulators of PI3K (24)(25)(26), such as pSHIP1, pSHIP2, and PTEN (Fig. 6B). Total protein expressions of the E3 ubiquitin ligase Cbl-b, a negative regulator of TCR signaling, and phosphatases PP2A and PHLPP, negative regulators of activated AKT (24)(25)(26), were also not affected by clozapine (Fig. 6B). In conclusion, costimulation of hM3Dq did not affect tyrosine phosphorylation of major TCR signaling events but greatly reduced the phosphorylation and by inference the activation of pAkt. AKT phosphorylates targets on Ser and Thr residues, primarily within a minimal consensus recognition motif of RXXS*/T* (where X is any amino acid and * denotes a preference for large hydrophobic residues) to often inhibit the function of the given target. AKT activation can promote cell survival, proliferation, growth, and changes in cellular metabolic pathways through its numerous downstream targets. Three best-established downstream targets of AKT include glycogen synthase kinase 3 (GSK-3), the forkhead box O transcription factor (FoxO), and tuberous sclerosis complex 2 (TSC2) (21). Consistent with decreased pAKT, clozapine also reduced pAKT substrates, including pGSK3b at pSer9, pFoxO1, and pTCS2 (Fig. 6C). In contrast, pGSK3b Y2176, which is not an AKT substrate, and phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), which phosphorylates AKT at T308, showed no substantial impact by clozapine treatment. Thus, stimulation of muscarinic receptors strongly reduced activation of pAKT pathways and its downstream molecules, which may explain the effect on IL-2 production in hM3Dq/β1T cells.

Inhibition of PI3K Reduces IL-2 Production in TCR-Stimulated
hM3Dq/β1 CD4 T Cells. Since the PI3K pathway is involved in AKT activation, we asked whether inhibition of PI3K would mimic the effect of muscarinic receptor stimulation in selectively blocking IL-2 but not IFNγ, CD69, or CD25 expression in TCR-stimulated cells. A PI3K inhibitor (Ly294002) was used to determine whether pAKT pathway is critical for IL-2 production. Indeed, we found that the PI3K inhibitor Ly294002 strongly reduced IL-2 expression in TCR or PMA plus ionomycinstimulated T cells that were preactivated via TCR stimulation followed by culture in IL-2 or IL-7 ( Fig. 7 A-H). In contrast, the PI3K inhibitor showed much less effect on IFN-γ, CD65, and CD25 expression compared to IL-2 expression in the preactivated T cells (Fig. 7 A-H). In conclusion, the PI3K/pAkt pathway is critical for IL-2 production in previously stimulated T cells.

Discussion
Studying the functions of muscarinic receptors, which are largely PTK independent, might inspire new therapies for bypassing states of T cell unresponsiveness, such as T cell anergy and T cell exhaustion, or the negative regulatory impact of coinhibitory receptors that depend on tyrosine phosphorylation pathways. However, we found that stimulation of muscarinic receptors M1 and hM3Dq showed much lower-than-anticipated calcium responses in primary T cells compared to those in Jurkat cells. Transduction of PLCβ1, a predominant PLCβ isoform expressed in Jurkat but not in primary T cells (13,15), in preactivated T cells led to increased muscarinic receptor and PLCβ1 expression and greatly amplified T cell calcium increases and phosphorylated ERK in response to stimulation of muscarinic receptors. Unexpectedly, whereas or phosphorylation of AP-1. We noted that the relatively selective effect on IL-2 may be attributable, in part, to a reduction in the 3′UTR activity of IL-2 mRNA that could lead to reduced IL-2 mRNA stability. How clozapine stimulation of the muscarinic HM3Dq receptor led to inhibition of IL-2 3′UTR was not entirely clear. However, we found that clozapine stimulation of hM3Dq/β1 T cells resulted in a reduction in pAKT and its downstream pathway signaling molecules. This could impact IL-2 production in hM3Dq/β1-stimulated T cells based on a negative regulatory effect on an inhibitor of the PI3K pathway. Indeed, we found that a potent PI3K inhibitor reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells. Together, these findings suggest that the pAKT pathway is critical for IL-2 production in previously activated T cells. Future studies in designing CAR T cells or other immune therapies might need to consider whether a PI3K/pAKT increase and subsequent IL-2 production by these cells would be desirable. We would predict, but have not studied here, the ability of hM3Dq receptor to resist the negative regulatory impact of ITIM and other inhibitory constraints targeting tyrosine phosphorylation pathways. Still, attention needs to consider situations where IL-2 production is important. The insights gained from these studies will be informative about the key aspects of pAKT and IL-2 production by T cells.
Previous studies have supported our results showing that the PI3K/pAKT pathway is critical for IL-2 induction by using the PI3K inhibitors LY294002, IC87114, and wortmannin (27,28). LY294002 and wortmannin blocked IL-2 production and T cell proliferation in antigen-stimulated or anti-CD3-stimulated T cells (27,28), thereby supporting a role for PI3K signaling in the IL-2 production. However, these PI3K inhibitors also reduced the production of other cytokines, including IFN-γ, IL-4, IL-17, and TNF-a in anti-CD3-stimulated naïve T cells (27,28). The different effects of LY294002 on IFN-γ production between previous studies and the effects of muscarinic receptors in this study were probably due to the two separate activation/differentiation states of T cells that were studied, i.e., naïve T cells versus previously stimulated T cells (preactivated via TCR stimulation followed by IL-2 or IL-7), respectively. These results suggest that IFN-γ production in previously stimulated T cells is less sensitive to PI3K inhibitors.
The impact of the PI3K/pAKT pathway in promoting IL-2 production was a bit unexpected based on classical studies of the IL-2 upstream promoter. However, more recent studies suggest that the PI3K/AKT pathway promotes IL-2 mRNA stability by mediating effects on RBPs. RBPs, which determine mRNA maturation, localization, stability, and translation, exhibit binding activity through the recognition of adenylate and uridylaterich elements (AREs) or U-rich elements that are often found in the 3′UTR of their target mRNAs (22,23). Stimulation of hM3Dq led to reduced 3′UTR activities of IL-2 mRNA and subsequently IL-2 mRNA and protein expression in TCR-stimulated hM3Dq/β1T cells. In contrast, 3′UTR activities of IFN-γ mRNA and protein expression were not affected by clozapine stimulation of TCR-stimulated hM3Dq/β1 T cells. Together, these results suggest that stimulation of hM3Dq influences specific subsets of RNA-binding proteins involved in regulating IL-2 mRNA stability. Binding of YB-1 and nucleolin to the 5′-UTR and binding of HuR and NF-90 to the 3′-UTR have been implicated in the PI3K/CD28-mediated stabilization of the IL-2 mRNA stability (20,(29)(30)(31)(32). Interestingly, HuR-deleted CD4+ T cells have strikingly increased IL-2, with no change in IFN-γ expression (33,34). Future studies should focus on characterizing these RNA-binding proteins, especially HuR, in influencing IL-2 but not IFN-γ mRNA stability in stimulated hM3Dq/β1 T cells. Such studies could impact the potential use of the muscarinic receptor approach described here in future immunotherapy.

Methods
Mice. Mice, 8 to 12 wk of age, of the strains C57BL/6, hM3Dq [B6N;129-Tg(CAG-CHRM3*, mCitrine)1Ute/J, Strain #:026220], and Lck-Cre [B6.Cg-Tg(Lckcre)548Jxm/J, Strain #:003802] were purchased from The Jackson Laboratory. All mice were housed in the specific pathogen-free facilities at the University of California, San Francisco, and were treated according to protocols that were approved by the UCSF Animal Care Ethics and Veterinary Committees, and in accordance with NIH guidelines. The inducible PLCβ1-mCherry mouse strain was generated by gene targeting in C57BL/6 ES cells similar to previous studies (35,36). PLCβ1-IRES-mCherry was cloned into the CTV plasmid (Addgene plasmid # 15912; http://n2t.net/ addgene:15912; RRID:Addgene_15912). The CAG-PLCβ1-mCherry allele allows Cre recombinase-inducible expression of a CAG promoter-driven PLCβ1-IRES-mCherry. Following Cre-mediated removal of an upstream floxed-STOP cassette, expression of PLCβ1 and mCherry fluorescent protein is observed. We successfully generated seven ES cell clones with properly inserted PLCβ1 in the Rosa 26 locus, confirmed by long-range PCR. We used PCR primers 5′ and 3′ of the Rosa26 arm of genomic DNA isolated to detect 2 representative ES clones (11 and 94) containing PLCβ1-IRES-mCherry knocked into the Rosa26 locus. PLCβ1 expression in T cells was monitored by the expression of mCherry separated from the PLCβ1 by an IRES sequence. Two ES clones (11 and 94) were selected to be injected into embryos to generate chimeric mice. Inducible PLCβ1 knock-in chimeras were crossed with hM3Dq × Lck-Cre mice to generate mice in which only T cells expressed both hM3Dq and PLCβ1 to study muscarinic receptor and PLCβ1 pathways in T cells.
For PLCβ1 genotyping, an mCherry forward primer GACCGCCAAGCTGAAGGT GACC and a reverse primer GCGCGTTCGTACTGTTCCACGA were used. The desired amplicon within mCherry is 525 bp. Inducible PLCβ1 knock-in mice were crossed with Lck-Cre × hM3Dq mice to generate LckCre-hM3Dq-PLCβ1 mice in which only T cells expressed both hM3Dq and PLCβ1 to study muscarinic receptor and PLCβ1 pathways in T cells.
Lentivirus Transduction. M1 or hM3Dq was cloned into the pHR backbone plasmid under the control of the EF1A promoter. A C-terminal P2A self-cleaving peptide followed by mCherry was incorporated to assess transduction efficiency and expression levels. The packaging vector pCMV dR8.91, envelope vector pMD 2.G, and pHR M1 or hM3Dq constructs were transiently cotransfected into LX-293T cells with TransIT-LT1 reagent (Mirus Bio Mio2300). Supernatants containing virus particles were collected 48 h after transfection, filtered, and pelleted before use. The virus particles were resuspended in PBS and stored at -80 °C. The supernatants were collected and filtered 48 h after transfection, and the Jurkat cells were transduced by centrifugation (1,200 g, 45 min). Retroviral Transduction. M1 or hM3Dq was cloned into the MSCV backbone plasmid [MSCV-IRES-GFP was a gift from Tannishtha Reya (Addgene, plasmid #20672; http://n2t.net/addgene:20672; RRID:Addgene_20672)]. A flag-tag sequence was added at the C terminus of M1 to monitor M1 expression level. PLCβ1 was cloned into the MSCV backbone plasmid at the EcoRI and XhoI sites [MSCV-IRES-mCherry FP was a gift from Dario Vignali (Addgene, plasmid #52114; http://n2t.net/addgene:52114; RRID:Addgene_52114)]. The pcL-Eco and MSCV vectors were cotransfected into Phoenix-Eco packaging cell line using Lipofectamine 2000 (Invitrogen). Supernatants containing virus particles were collected 48 h and 72 h after transfection. The virus particles were concentrated using a Retro-X concentrator (Takara, Cat 631455) resuspended in PBS and stored at -80 °C. Retroviral transduction of T cells were stimulated with plate-bound anti-CD3 (2C11, 1 μg/mL), soluble anti-CD28 (37.51, 2 μg/mL), and IL-2 (40 U/mL) in culture medium (RPMI supplemented with L-glutamine/streptomycin and 10% fetal bovine serum) in 96-well round-bottom plates at 37 °C in 5% CO2 in a 6-well plate for 36 h. Lipofectamine was added to the thawed retroviral supernatant at a concentration of 8 μg/mL and incubated for 30 min at room temperature. Prestimulated T cells were transduced with the retroviral supernatant containing Lipofectamine by centrifugation (1,200 g, 25 min). Two days following transduction, viral supernatant was replaced with culture media containing IL-2 (40 U/mL). T cells were cultured with IL-2 for at least two additional days.
Statistical Analysis. All data were displayed as mean ± SD. Statistical analysis was done using an unpaired two-tailed Student's t test. A P value < 0.05 was considered significant. Data, Materials, and Software Availability. All study data are included in the article and/or SI Appendix.